ARF and p53 coordinate tumor suppression of an oncogenic IFN-β-STAT1-ISG15 signaling axis

SummaryThe ARF and p53 tumor suppressors are thought to act in a linear pathway to prevent cellular transformation in response to various oncogenic signals. Here, we show that loss of p53 leads to an increase in ARF protein levels, which function to limit the proliferation and tumorigenicity of p53-deficient cells by inhibiting an IFN-β-STAT1-ISG15 signaling axis. Human triple-negative breast cancer (TNBC) tumor samples with coinactivation of p53 and ARF exhibit high expression of both STAT1 and ISG15, and TNBC cell lines are sensitive to STAT1 depletion. We propose that loss of p53 function and subsequent ARF induction creates a selective pressure to inactivate ARF and propose that tumors harboring coinactivation of ARF and p53 would benefit from therapies targeted against STAT1 and ISG15 activation

Cathepsin K-Cre causes unexpected germline deletion of genes in mice.

Osteoclasts are terminally differentiated cells that attach to bone and secrete proteases to degrade the bone matrix. The primary protease responsible for the degradation of the organic component of the bone matrix is Cathepsin K, which was largely thought to be unique to osteoclasts. Given its apparent selective expression in osteoclasts, the Cathepsin K promoter has been engineered to drive the expression of Cre recombinase in mice and has been the most relevant tool for generating osteoclast-specific gene loss. In an effort to understand the role of the ARF tumor suppressor in osteoclasts, we crossed Arf (fl/fl) mice to Ctsk(Cre/+) mice, which unexpectedly resulted in the germline loss of Arf. We subsequently confirmed Cre activity in gametes by generating Ctsk(Cre/+); Rosa(+) mice. These results raise significant concerns regarding in vivo bone phenotypes created using Ctsk(Cre/+) mice and warrant further investigation into the role of Cathepsin K in gametes as well as alternative tools for studying osteoclast-specific gene loss in vivo

Cre expression in <i>Ctsk^Cre/+</i> mouse gametes is verified with <i>Rosa⁺</i> mice.

<p>Reproductive organs of indicated genotypes were analyzed for Cre activity by LacZ staining. (A) In ovaries of <i>Rosa⁺</i>; <i>Ctsk^Cre/+</i> mice, Cre activity was detected in oocytes and cells surrounding the developing oocytes (left panel). Controls were negative for LacZ staining (right panel). Scale bar = 50 µM. (B) In testes of <i>Rosa⁺</i>; <i>Ctsk^Cre/+</i> mice, Cre activity was detected primarily in spermatozoa (left panel). Controls were negative for LacZ staining (right panel). Scale bar = 100 µM.</p

Crossing <i>Arf^fl/fl</i> mice with <i>CtsK^Cre/+</i> mice results in germline <i>Arf</i> loss.

<p>(A) Three sets of primer pairs were designed to detect the presence of floxed <i>exon 1beta</i>, which is unique to <i>Arf.</i> (B) PCR products were not detected upon genotyping new mice for the presence of the 5′ loxP site (far left panel). This cannot be attributed to an absence of DNA (middle left panel). Controls are mice that were never crossed with <i>Ctsk^Cre/+</i> mice. Two sets of primer pairs were used to detect the presence of <i>exon 1beta</i>. Each set detects a product indicating loss of <i>exon 1beta</i> (product size labeled as “KO” in table). (C) Immunofluorescent staining for ARF in testis tissues indicates the loss of <i>exon 1beta</i> at the protein level (scale bar = 100 µM).</p

Crossing <i>Arf^fl/fl</i> mice with <i>CtsK^Cre/+</i> mice results in spontaneous tumor formation.

<p>(A) <i>Ctsk^Cre/+</i>; <i>Arf^fl/fl</i> mice display both fibrosarcomas (top image) and lymphomas with splenomegaly (middle and bottom images). (B) Spontaneous tumor development occurred in all <i>Ctsk^Cre/+</i>; <i>Arf^fl/fl</i> mice.</p

Cre is expressed in the gametes of <i>Ctsk^Cre/+</i> mice.

<p>(A) Real-time PCR was used to quantify the presence of Cathepsin K mRNA (blue) and Cre mRNA (red) in ovary (left) and testis (right) for all indicated genotypes (n = 3). Data are represented as means ± SD. A two-tailed t-test was used to generate indicated p values. (B) Ovaries from WT mice analyzed by IHC for Cathepsin K. Top panels, scale bar = 200 µM. Bottom, left panel shows staining without primary antibody. An adjacent section to the control (bottom, middle) was incubated with primary antibody (scale bar = 100 µM). Red box indicates positively-stained oocyte (bottom, right scale bar = 20 µM).</p

ARF tumor suppression in the nucleolus

AbstractSince its discovery close to twenty years ago, the ARF tumor suppressor has played a pivotal role in the field of cancer biology. Elucidating ARF's basal physiological function in the cell has been the focal interest of numerous laboratories throughout the world for many years. Our current understanding of ARF is constantly evolving to include novel frameworks for conceptualizing the regulation of this critical tumor suppressor. As a result of this complexity, there is great need to broaden our understanding of the intricacies governing the biology of the ARF tumor suppressor. The ARF tumor suppressor is a key sensor of signals that instruct a cell to grow and proliferate and is appropriately localized in nucleoli to limit these processes. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease